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ocram's Content

There have been 2 items by ocram (Search limited from 25-September 19)

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#178727 Lysis of osteoblastic cell lines for dual luciferase assay

Posted by ocram on 31 October 2016 - 03:51 AM in Cell Biology

I need to perform a dual luciferase assay on transfected MC3T3 cells, an osteoblastic cell line.  I have already performed this assay successfully on Cos7 cells, but I want to transfer this same assay to an osteoblastic cell line.  My problem lies in the cell lysis step, following 48h  transfections.  I have used various osteoblastic cell lines, but come up consistently with the same problem.  Since the cells form a collagenous matrix around them on the culture dish they are very difficult to lyse for the assay.  The assay  (Promega Dual Luciferase Assay) requires passive lysis, with the kit specific lysis buffer, but this buffer merely detatches the cells  and then clumps them together without lysing them.  I have tried treating the cells with collagenase (Dispase ) prior to lysis and then removing the collagenase.  The dispase works, but as soon as I add the lysis buffer the cells clump together again.  I am at a loss as to what to do,  since I need to eventually perform my experiment in a 48 or 96 well format, so I need to be sure that the lysis has worked!

#177071 consistent expression of overlarge proteins

Posted by ocram on 02 November 2015 - 08:00 AM in Protein Expression and Purification

I have recently joined a new lab and started to express bacterial recombinant proteins again.  I have expressed three, all in the same vector, a modified pET vector with simple His tag in BL21(DE3) and all three proteins are too large, by between 5 -15kDa.  The sequences are fine, (both of the empty vector and recombinants) their mobility is not affected by running on Tris-glycine or Tricine gels, but I am able to purify them on HisTrap and they give a signal with anti-His monoclonal on Western.  The problem is I am not able to further purify them either by IEX or size exclusion and their size bothers me.  How can I be sure these are really my proteins - any suggestions..??

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